The expression of the erythropoietin receptor (EpoR) on the surface of erythroid progenitors is necessary for the binding of erythropoietin (Epo), the primary stimulator of erythropoiesis. We examined control elements within the promoter of the human erythropoietin receptor (hEpoR) gene which contains binding sites for transcription factors, GATA-1 with erythroid specificity and Sp1, but no TATA sequences. In the hEpoR proximal promoter region we have observed binding of nuclear proteins to the GATA-1, Sp1 and AP2 binding consensus sequences, and a region around -175 bp. Regions containing the AP2 binding site and around - 175 provided positive and negative transcription regulation, respectively. The GATA-1 binding site contributed to positive regulation in erythroid cells only. Although the endogenous levels of GATA-1 in K562 and OClM1 cells were comparable, cotransfection of the hEpoR promoter with a GATA-1 expression vector to further increase GATA-1 levels increased hEpoR promoter activity in K562 cells reaching a five fold increase at saturation, but exhibited no effect in OClM1 cells. In non-erythroid cells, hEpoR promoter activity was increased by co- transfection with GATA-1 and to a lesser extent by mutation and inversion of the GATA-1 binding site. GATA-1 provides negative regulation of the hEpoR promoter in non-erythroid cells. Promoter activity was still present with deletion of the GATA-1 binding site leaving the Sp1 binding site. Although mutation of the Sp1 binding site markedly decreased promoter activity, the promoter could still be transactivated by GATA-1 Hence, as with other TATA-less promoters, Sp1 alone can activate transactivate, indicating that Sp1 is not required and may not interact directly with GATA-1 for hEpoR transactivation. The role of the Sp1 and GATA-1 binding sites in the hEpoR promoter suggest that interactions of Sp1 and GATA-1 with the hEpoR gene contribute significantly to the differential transcription observed for hEpoR tissue and state specific expression.